Chondroitin polymerase and dna encoding the same

ABSTRACT

A chondroitin polymerase having such properties that it transfers GlcUA and GalNAc alternately to a non-reduced terminal of a sugar chain from a GlcUA donor and a GalNAc donor, respectively, and the like; and a process for producing the chondroitin polymerase.

BACKGROUND OF THE PRESENT INVENTION

1. Field of the Present Invention

The present invention relates to a novel chondroitin polymerase(chondroitin synthase), a DNA encoding the same, a method for producingthe chondroitin polymerase, a method for producing a sugar chain havingthe disaccharide repeating unit of chondroitin, a hybridization probefor the chondroitin polymerase and the like.

2. Brief Description of the Background Art

First, abbreviations commonly used in the present specification aredescribed.

In the formulae and the like, “GlcUA”, “GalNAc”, “GlcNAc”, “UDP” and “-”represent D-glucuronic acid, N-acetyl-D-galactosamine,N-acetyl-D-glucosamine, uridine 5′-diphosphate and a glycosidic linkage,respectively.

Chondroitin is a sugar chain comprised of a disaccharide repeatingstructure of GlcUA residue and GalNAc residue(-GlcUAβ(1-3)-GalNAcβ(1-4)-; hereinafter also referred to as“chondroitin backbone”), and a sugar chain in which the chondroitin isfurther sulfated is chondroitin sulfate.

Regarding an enzyme which synthesizes chondroitin from a GlcUA donor anda GalNAc donor by alternately transferring GlcUA and GalNAc to anacceptor (chondroitin polymerase or chondroitin synthase) and DNA whichencodes the same, only a Pasteurella multocida chondroitin synthase (J.Biol. Chem., 275 (31), 24124-24129 (2000)) is known.

Also, certain Escherichia coli strain (Escherichia coli serotype 05:K4(L):H4, hereinafter referred to as “Escherichia coli strain K4”)produces a polysaccharide having a chondroitin backbone, as a capsularantigen, but its structure is a trisaccharide repeating structure inwhich fructose is linked to a side chain of the GlcUA residue at theβ2-3 position. Accordingly, it was unclear whether the Escherichia colistrain K4 really has a chondroitin polymerase as its own capsularantigen synthesizing system.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a novel chondroitinpolymerase, a DNA encoding the same, a process for producing thechondroitin polymerase, a process for producing a sugar chain having thedisaccharide repeating unit of chondroitin, a hybridization probe forthe chondroitin polymerase and the like.

This and other objects of the present invention have been accomplishedby a novel chondroitin polymerase having specific properties describedbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a restriction map of λ phage clones which contain a part ofR-I region or R-III region of the K antigen gene cluster of Escherichiacoli strain K4 .

FIG. 2 shows the open reading frame (ORF) of the R-II region of the Kantigen gene cluster of Escherichia coli strain K4 .

FIG. 3 is a graph showing transfer of GalNAc to hexasaccharide ofchondroitin sulfate C by the enzyme of the present invention.

FIG. 4 is a graph showing transfer of GalNAc to hexasaccharide ofchondroitin sulfate C by the enzyme of the present invention and thesizes of the produced sugar chain.

FIG. 5 is a graph showing transfer of each monosaccharide whenUDP-GlcUA, UDP-GlcNAc or UDP-GalNAc was used as the donor, andhexasaccharide or heptasaccharide of chondroitin sulfate C was used asthe acceptor.

FIG. 6 is a graph showing the influence of temperature on the activityof the enzyme of the present invention.

FIG. 7 is a graph showing gel filtration patterns of enzyme reactionproducts after various enzyme reaction times.

FIG. 8 is a graph showing the relationship between the enzyme reactiontime and the incorporation amount of radioactivity.

FIG. 9 is a graph showing the relationship between the incorporatedradioactivity (V) and the substrate concentration of UDP-sugar (S).

FIG. 10 is a graph showing double reciprocal plots.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have conducted intensive studies and found a novelchondroitin polymerase produced by specific microorganism (Escherichiacoli strain K4 (Escherichia coli serotype 05:K4 (L):H4, ATCC 23502)),isolated cDNA encoding the chondroitin polymerase, and succeeded inpreparing the chondroitin polymerase using the cDNA. Thus, the presentinvention has been completed.

Also, this and other objects of the present invention have beenaccomplished by a process for producing the chondroitin polymerase byisolating cDNA encoding the chondroitin polymerase and using the cDNA.The term “chondroitin synthesis” or “synthesis of chondroitin” as usedherein is a concept which includes elongation of the sugar chain ofchondroitin by transferring and adding monosaccharides to a sugar chainsuch as chondroitin. Accordingly, the reaction for elongating the sugarchain of chondroitin by alternately transferring and adding thechondroitin synthesizing monosaccharides (GlcUA and GalNAc) to the sugarchain is included in a concept of “chondroitin synthesis” or “synthesisof chondroitin”.

The present invention relates to a chondroitin polymerase (hereinafteralso referred to as “the enzyme of the present invention”) having thefollowing properties:

(1) Action:

the polymerase transfers GlcUA and GalNAc alternately to a non-reducedterminal of a sugar chain from, a GlcUA donor and a GalNAc donor,respectively;

(2) Substrate Specificity:

the polymerase transfers GlcUA to an oligosaccharide having GalNAc onits non-reduced terminal and a chondroitin backbone from a GlcUA donor,but does not substantially transfer GalNAc to the oligosaccharide from aGalNAc donor;

the polymerase transfers GalNAc to an oligosaccharide having GlcUA onits non-reduced terminal and a chondroitin backbone from a GalNAc donor,but does not substantially transfer GlcUA to the oligosaccharide from aGlcUA donor;

(3) Influence by Metal Ions and the Like:

the polymerase acts in the presence of Mn²⁺ ion but does notsubstantially act in the presence of Ca²⁺ ion, Cu²⁺ ion orethylenediaminetetraacetic acid.

The enzyme of the present invention is preferably derived fromEscherichia coli.

The present invention relates to a protein selected from the groupconsisting of the following (A) and (B) (hereinafter also referred to as“the protein of the present invention”):

(A) a protein comprising the amino acid sequence represented by SEQ IDNO:2;(B) a protein comprising the amino acid sequence in which one or a fewamino acid residue(s) in the amino acid sequence represented by SEQ IDNO: 2 are deleted, substituted, inserted or transposed, and having achondroitin polymerase activity.

The present invention relates to a DNA comprising any one of thefollowing (a) to (c) (hereinafter also referred to as “the DNA of thepresent invention”):

(a) a DNA which encodes a protein consisting of the amino acid sequencerepresented by SEQ ID NO: 2;(b) a DNA which encodes a protein consisting of an amino acid sequencein which one or a few amino acid residue(s) in the amino acid sequencerepresented by SEQ ID NO: 2 are deleted, substituted, inserted ortransposed, and having a chondroitin polymerase activity;(c) a DNA which hybridizes with

(i) the DNA in (a),

(ii) a DNA complementary to the DNA in (a), or

(iii) a DNA having a part of nucleotide sequences of the DNA in (i) and(ii) under stringent conditions.

The DNA in (a) is preferably represented by SEQ ID NO: 1.

The present invention relates to a vector comprising the DNA of thepresent invention (hereinafter also referred to as “the vector of thepresent invention”).

The vector of the present invention is preferably an expression vector.

The present invention relates to a transformant in which a host istransformed with the vector of the present invention (hereinafter alsoreferred to as “the transformant of the present invention”).

The present invention relates to a process for producing a chondroitinpolymerase, which comprises: growing the transformant of the presentinvention; and collecting the chondroitin polymerase from the grownmaterial (hereinafter also referred to as “the enzyme production processof the present invention”).

The present invention relates to a sugar chain synthesizing agent,comprising an enzyme protein which comprises an amino acid sequencerepresented by the following (A) or (B) and has enzymic activities ofthe following (i) and (ii) (hereinafter also referred to as “thesynthesizing agent of the present invention”):

(A) the amino acid sequence represented by SEQ ID NO:2 ;(B) an amino acid sequence in which one or a few amino acid residue(s)in the amino acid sequence represented by SEQ ID NO:2 are deleted,substituted, inserted or transposed;(i) GlcUA and GalNAc are alternately transferred to a non-reducedterminal of a sugar chain from a GlcUA donor and a GalNAc donor,respectively;(ii) GlcNAc is transferred to a non-reduced terminal of a sugar chainhaving GlcUA on the non-reduced terminal from a GlcNAc donor.

The present invention relates to a process for producing a sugar chainrepresented by the following formula (3), which comprises at least astep of allowing the synthesizing agent of the present invention tocontact with a GalNAc donor and a sugar chain represented by thefollowing formula (1) (hereinafter also referred to as “the sugar chainproduction process 1 of the present invention”):

GlcUA-X—R¹  (1)

GalNAc-GlcUA-X—R¹  (3)

wherein X represents GalNAc or GlcNAc; R¹ represents an any group; andother symbols have the same meanings as described above.

The present invention relates to a process for producing a sugar chainrepresented by the following formula (4), which comprises at least astep of allowing the synthesizing agent of the present invention tocontact with a GlcNAc donor and a sugar chain represented by thefollowing formula (1) (hereinafter referred to as “the sugar chainproduction process 2 of the present invention”):

GlcUA-X—R¹  (1)

GlcNAc-GlcUA-X—R¹  (4)

wherein all symbols have the same meanings as described above.

The present invention relates to a process for producing a sugar chainrepresented by the following formula (5), which comprises at least astep of allowing the synthesizing agent of the present invention tocontact with a GlcUA donor and a sugar chain represented by thefollowing formula (2) (hereinafter also referred to as “the sugar chainproduction process 3 of the present invention”):

GalNAc-GlcUA-R²  (2)

GlcUA-GalNAc-GlcUA-R²  (5)

wherein R² represents an any group; and other symbols have the samemeanings as described above.

The present invention relates to a process for producing a sugar chainselected from the following formulae (6) and (8), which comprises atleast a step of allowing the synthesizing agent of the present inventionto contact with a GalNAc donor, a GlcUA donor and a sugar chainrepresented by the following formula (1) (hereinafter also referred toas “the sugar chain production process 4of the present invention”):

GlcUA-X—R¹  (1)

(GlcUA-GalNAc)n-GlcUA-X—R¹  (6)

GalNAc-(GlcUA-GalNAc)n-GlcUA-X—R¹  (8)

wherein n is an integer of 1 or more, and other symbols have the samemeanings as described above.

The present invention relates to a process for producing a sugar chainselected from the following formulae (7) and (9), which comprises atleast a step of allowing the synthesizing agent of the present inventionto contact with a GalNAc donor, a GlcUA donor and a sugar chainrepresented by the following formula (2) (hereinafter also referred toas “the sugar chain production process 5of the present invention”):

GalNAc-GlcUA-R²  (2)

(GalNAc-GlcUA)n-GalNAc-GlcUA-R²  (7)

GlcUA-(GalNAc-GlcUA)n-GalNAc-GlcUA-R²  (9)

wherein all symbols have the same meanings as described above.

The present invention relates to a hybridization probe comprising anucleotide sequence complementary to the nucleotide sequence representedby SEQ ID NO:1 or a part thereof (hereinafter also referred to as “theprobe of the present invention”).

The present invention relates to a glycosyltransfer catalyst(hereinafter also referred to as “the catalyst of the presentinvention”) which comprises an enzyme protein comprising an amino acidsequence selected from the following (A) and (B), and is capable oftransferring GlcUA, GalNAc and GlcNAc to a non-reduced terminal of asugar chain from a GlcUA donor, a GalNAc donor and a GlcNAc donor,respectively:

(A) the amino acid sequence represented by SEQ ID NO: 2;(B) an amino acid sequence in which one or a few amino acid residue(s)in the amino acid sequence represented by SEQ ID NO:2 are deleted,substituted, inserted or transposed.

The present invention is explained below in more detail.

<1> Enzyme of the Present Invention and Protein of the Present Invention

The enzyme of the present invention is a chondroitin polymerase havingthe following properties (1) to (3).

(1) Action:

The enzyme of the present invention transfers GlcUA and GalNAcalternately to a non-reduced terminal of a sugar chain from a GlcUAdonor and a GalNAc donor, respectively.

As the GlcUA donor, a nucleoside diphosphate-GlcUA is preferred, andUDP-GlcUA is particularly preferred. Also, as the GalNAc donor, anucleoside diphosphate-GalNAc is preferred, and UDP-GalNAc isparticularly preferred.

The enzyme of the present invention transfers GlcUA and GalNAcalternately to a non-reduced terminal of a sugar chain (acceptor) fromthese respective saccharide donors. For example, when GlcUA is firsttransferred to a non-reduced terminal of a sugar chain (acceptor),monosaccharides are transferred in such a manner that GalNAc is thentransferred, GlcUA is then transferred, GalNAc is then transferred andso on. In the same manner, when GalNAc is first transferred to thenon-reduced terminal of a sugar chain (acceptor), monosaccharides aretransferred in such a manner that GlcUA is then transferred, GalNAc isthen transferred, GlcUA is then transferred and so on. As a result, adisaccharide repeating structure of GlcUA residue and GalNAc residue,namely a chondroitin backbone, is synthesized by the enzyme of thepresent invention.

As the sugar chain which becomes the acceptor of monosaccharides, asugar chain having a chondroitin backbone is preferable. As the sugarchain having a chondroitin backbone, chondroitin sulfate and chondroitincan be exemplified. Among chondroitin sulfates, a chondroitin sulfatewhich is mainly comprising a chondroitin 6-sulfate structure and alsocontains a small amount of chondroitin 4-sulfate structure (hereinafterreferred to as “chondroitin sulfate C”) is preferable.

Also, the sugar chain which becomes an acceptor is more preferably anoligosaccharide. The size of the oligosaccharide is not particularlylimited, but when the acceptor is an oligosaccharide of chondroitinsulfate C, hexasaccharide or heptasaccharide is preferable, andtetra-saccharide or hexasaccharide is preferable when it is anoligosaccharide of chondroitin.

Also, it is preferable that the enzyme of the present invention iscapable of further transferring GalNAc to a sugar chain having ahyaluronic acid backbone (a disaccharide repeating structure of GlcUAresidue and GlcNAc residue) from a GalNAc donor. It is preferable thatthe sugar chain having a hyaluronic acid backbone is also anoligosaccharide. The size of the oligosaccharide is not particularlylimited, but those which are composed of about hexasaccharides areparticularly preferable.

(2) Substrate Specificity:

The enzyme of the present invention transfers GlcUA to anoligosaccharide having GalNAc on its non-reduced terminal and achondroitin backbone from a GlcUA donor, but does not substantiallytransfer GalNAc to the oligosaccharide from a GalNAc donor.

The enzyme of the present invention transfers GalNAc to anoligosaccharide having GlcUA on its non-reduced terminal and achondroitin backbone from a GalNAc donor, but does not substantiallytransfer GlcUA to the oligosaccharide from a GlcUA donor.

It is preferable that the enzyme of the present invention which furtherdoes not substantially transfer GlcNAc from a GlcNAc donor to anoligosaccharide having GalNAc on its non-reduced terminal and also has achondroitin backbone. Furthermore, it is preferable that the enzyme ofthe present invention further transfers GlcNAc from a GlcNAc donor to anoligosaccharide having GlcUA on its non-reduced terminal and also has achondroitin backbone. However, it is preferable that GlcUA is notsubstantially transferred from a GlcUA donor to an oligosaccharideproduced by the transfer of GlcNAc.

Also, it is preferable that the enzyme of the present inventiontransfers GalNAc from a GalNAc donor to an oligosaccharide having GlcUAon its non-reduced terminal and also having a hyaluronic acid backbone,but does not substantially transfer GlcUA from a GlcUA donor.

(3) Influence by Metal Ions and the Like:

The enzyme of the present invention acts in the presence of Mn²⁺ ion butdoes not substantially act in the presence of Ca²⁺ ion, Cu²⁺ ion orethylenediaminetetraacetic acid.

Also, it is preferable that the enzyme of the present invention furtheracts in the presence of Fe²⁺ or Mg²⁺ ion. Moreover, it is preferablethat the degree of action (enzyme activity) of the enzyme of the presentinvention in the presence of Mn²⁺ ion is higher than its degree ofaction (enzyme activity) in the presence of Fe²⁺ or Mg²⁺ ion.

Also, it was observed that when a reaction was carried out using theenzyme of the present invention at a temperature of 25° C. or more, sizeof the reaction product (chondroitin chain) became small as the reactiontemperature increased (cf., Examples shown below). Accordingly, it isconsidered that enzyme activity of the enzyme of the present inventiondecreases as the reaction temperature increases at 25° C. or more underthe reaction conditions described in the following Examples.

It is preferable that the enzyme of the present invention is derivedfrom Escherichia coli. Particularly, Escherichia coli strain having agene related to the production of capsular polysaccharide is preferable,and Escherichia coli strain whose capsular antigen (K) is “K4” is morepreferable.

As the Escherichia coli strain whose capsular antigen serotype is “K4 ”,Escherichia coli strain K4 (Escherichia coli serotype 05:K4 (L):H4) canbe preferably exemplified, and more specifically, ATCC 23502, NCDCU1-41, Freiburg collection number 2616 and the like can be preferablyexemplified.

It is preferable also that the enzyme of the present invention is aprotein selected from the following (A) and (B):

(A) a protein comprising the amino acid sequence represented by SEQ IDNO:2;(B) a protein comprising an amino acid sequence in which one or a fewamino acid residue(s) in the amino acid sequence represented by SEQ IDNO: 2 are deleted, substituted, inserted or transposed, and having achondroitin polymerase activity.

Although mutation such as substitution, deletion, insertion,transposition or the like can occur in amino acid sequences of proteinsexisting in the nature caused by the modification reactions inside thecells or during purification of proteins after their formation, inaddition to polymorphism and mutation of DNA molecules encoding them, itis known that certain proteins having such mutations show physiologicaland biological activities which are substantially identical to thecorresponding proteins having no mutations. Such proteins which haveslight structural differences but no significant differences in theirfunctions are also included in the protein of the present invention. Acase in which the above mutation is artificially introduced into theamino acid sequence of protein is the same. In such a case, it ispossible to prepare larger varieties of mutants. For example, it isknown that a polypeptide prepared by substituting a cysteine residue inthe amino acid sequence of human interleukin 2 (IL-2) by a serineresidue maintains the interleukin 2 activity (Science, 224, 1431(1984)). Also, it is known that a certain protein has a peptide regionwhich is not essential for its activity. For example, a signal peptideexisting in a protein which is secreted into the extracellular moietyand a pro-sequence which can be found in a protease precursor or thelike correspond to this case, and most of these regions are removedafter translation of proteins or during their conversion into activeproteins. Such proteins are proteins which are present in differentforms in terms of their primary structure but finally have similarfunctions.

The term “a few amino acid residues” as used herein means the number ofamino acids which may cause mutation in such a degree that activity ofthe chondroitin polymerase is not lost, and in the case of a proteincomposed of 600 amino acid residues for example, it means the number ofapproximately from 2 to 30, preferably from 2 to 15, and more preferablyfrom 2 to 8 .

Also, the protein of the present invention may contain an amino acidsequence of other protein or peptide, so long as it contains the aminoacid sequence of the above (A) or (B). That is, the protein of thepresent invention may be a fusion protein with other protein or peptide.

For example, fusion proteins of a protein comprising the amino acidsequence described in the above (A) or (B) with a marker peptide and thelike are also included in the protein of the present invention. Suchproteins of the present invention have a merit in that theirpurification can be carried out easily. Examples of the above markerpeptide include protein A, insulin signal sequence, His, FLAG, CBP(calmodulin-binding protein), GST (glutathione S-transferase) and thelike. For example, its fusion protein with protein A can be purifiedconveniently by affinity chromatography using an IgG-linked solid phase.In the same manner, a solid phase to which magnetic nickel is linked canbe used for a fusion protein with His tag, and a solid phase to which ananti-FLAG antibody is linked can be used for a fusion protein with FLAG.Also, since a fusion protein with insulin signal is secreted into anextracellular moiety (a medium or the like), extraction operations suchas cell disintegration and the like become unnecessary. It is preferablethat the protein of the present invention (the enzyme of the presentinvention) is soluble.

Preferred examples include a fusion protein with a peptide (His tag)represented by the amino acid sequence represented by SEQ ID NO:11. Itis preferable to carry out fusion of this His tag continuously at aposition just before the amino acid sequence represented by SEQ ID NO:2.The fusion protein can be produced by expressing a DNA in which thenucleotide sequence represented by SEQ ID NO:4 is connected continuouslyto a position just before the nucleotide sequence represented by SEQ IDNO:1. The fusion protein is soluble.

The “chondroitin polymerase activity” can be detected in accordance witha generally used glycosyltransferase assay method. Specifically, it canbe detected as the activity to synthesize chondroitin by transferringGlcUA and GalNAc alternately to a non-reduced terminal of a sugar chain(acceptor), using a GlcUA donor, a GalNAc donor and a sugar chain whichbecomes the acceptor.

For example, when GlcUA is transferred from a GlcUA donor to a sugarchain having GalNAc on its non-reduced terminal, and GalNAc istransferred from a GalNAc donor to a sugar chain having GlcUA on itsnon-reduced terminal, it can be judged that it has an activity totransfer GlcUA and GalNAc alternately to a non-reduced terminal of thesugar chain, namely the chondroitin polymerase activity. It ispreferable to employ the enzyme activity measuring method described inthe following Examples. By such a method, deletion, substitution,insertion or transposition of amino acids keeping the chondroitinpolymerase activity can be selected easily.

Production processes of the enzyme of the present invention and proteinof the present invention are not particularly limited, and they can beproduced by expressing the DNA of the present invention described belowin appropriate cells. Also, those which are isolated from naturalresources and produced by chemical synthesis and the like are includedin the enzyme of the present invention and the protein of the presentinvention as a matter of course. The production processes of the enzymeof the present invention (the protein of the present invention) usingthe DNA of the present invention will be described later.

<2> DNA of the Present Invention

The DNA of the present invention is a DNA comprising any one of thefollowing (a) to (c):

(a) a DNA encoding a protein consisting of the amino acid sequencerepresented by SEQ ID NO: 2;(b) a DNA encoding a protein consisting of an amino acid sequence inwhich one or a few amino acid residue (s) in the amino acid sequencerepresented by SEQ ID NO: 2 are deleted, substituted, inserted ortransposed, and having a chondroitin polymerase activity;(c) a DNA which hybridizes with the DNA in (a), a DNA complementary tothe DNA in (a) or a DNA having a part of nucleotide sequences of the DNAin (i) and (ii) under stringent conditions.

The DNA is preferably represented by SEQ ID NO:1.

The “stringent conditions” as used herein mean conditions under which aso-called specific hybrid is formed but a non-specific hybrid is notformed (cf. Sambrook, J. et al., Molecular Cloning, A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press (1989) and thelike). Examples of the “stringent conditions” include conditions inwhich the hybridization is carried out at 42° C. in a solutioncontaining 50% formamide, 4×SSC, 50 mM HEPES (pH 7.0), 10×Denhardt'ssolution and 100 μg/ml sermon sperm DNA, and the product is washed with2×SSC, 0.1% SDS solution at room temperature and then with 0.1×SSC, 0.1%SDS solution at 50° C.

The DNA of the present invention is originally obtained from Escherichiacoli strain having K4 antigen, but DNA samples obtained from othertransformed organism species and produced by chemical synthesis and thelike are also included therein as a matter of course. Productionprocesses of the DNA of the present invention are not particularlylimited too, but it is preferable to use, e.g., the process described inthe following Examples.

It is easily understood by those skilled in the art that DNAs havingvarious different nucleotide sequences due to degeneracy of genetic codeare present as the DNA of the present invention.

<3> Vector of the Present Invention

The vector of the present invention is a vector comprising the DNA ofthe present invention. Preferable DNA of the present invention in thevector of the present invention is the same as described in the above<2>. Also, since the vector of the present invention is preferably usedin the enzyme production process of the present invention which will bedescribed later, it is preferably an expression vector.

The vector of the present invention can be prepared by inserting the DNAof the present invention into a known vector.

As the vector into which the DNA of the present invention is inserted,for example, an appropriate vector which can express the introduced DNA(a phage vector, plasmid vector or the like) can be used, and it can beoptionally selected in response to each host cell into which the vectorof the present invention is inserted. Examples of the host-vector systeminclude a combination of a mammal cell such as COS cell, 3 LL-HK46 orthe like with an expression vector for mammal cell such as pGIR201(Kitagawa, H. and Paulson, J. C., J. Biol. Chem., 269, 1394-1401(1994)), pEF-BOS (Mizushima, S. and Nagata, S. Nucleic Acid Res., 18,5322 (1990)), pCXN2 (Niwa, H., Yamamura, K. and Miyazaki, J. Gene, 108,193-200 (1991)), pCMV-2 (manufactured by Eastman Kodak), pCEV18, pME18 S(Maruyama et al., Med. Immunol., 20, 27 (1990)), pSVL (manufactured byPharmacia Biotech) or the like and a combination of Escherichia coliwith a expression vector for procaryotic cell such as pTrcHis(manufactured by Invitrogen), pGEX (manufactured by Pharmacia Biotech),pTrc99 (manufactured by Pharmacia Biotech), pKK233-3 (manufactured byPharmacia Biotech), pEZZZ18 (manufactured by Pharmacia Biotech), pCH110(manufactured by Pharmacia Biotech), pET (manufactured by Stratagene),pBAD (manufactured by Invitrogen), pRSET (manufactured by Invitrogen),pSE420 (manufactured by Invitrogen) or the like. Additionally, an insectcell, yeast, Bacillus subtilis and the like can also be exemplified asthe host cell and various vectors corresponding thereto can beexemplified. Among the above host-vector systems, a combination ofEscherichia coli with pTrcHis is preferable.

Also, as the vector into which the DNA of the present invention isinserted, a vector constructed in such a manner that it expresses afusion protein of the protein of the present invention (enzyme of thepresent invention) with a marker peptide can also be used, which, asdescribed in the above <1>, is particularly preferable when thechondroitin polymerase expressed using the vector of the presentinvention is purified. Specifically, a vector comprising aHis-expressing nucleotide sequence (e.g., the nucleotide sequencerepresented by SEQ ID NO:4) is preferable.

When any of the above vectors is used, the DNA of the present inventioncan be connected with the vector after treating both of them withrestriction enzymes and the like and optionally carrying outsmooth-ending and connection of a cohesive end so that connection of theDNA of the present invention with the vector becomes possible.

As the process for producing the vector of the present invention, forexample, the process described in the following Examples can be used andis preferable.

<4> Transformant of the Present Invention

The transformant of the present invention is a transformant in which ahost is transformed with the vector of the present invention.

The “host” as used herein may be any host in which recombination by thevector of the present invention can be carried out but is preferably onewhich can exert function of the DNA of the present invention or arecombinant vector into which the DNA of the present invention isinserted. Examples of the host include animal cells, plant cells andmicrobial cells are included, and mammal cells such as COS cells (COS-1cell, COS-7 cells and the like), 3 LL-HK46 cell, etc., Escherichia coli,insect cells, yeast, Bacillus subtilis and the like. The host can beoptionally selected in response to each vector of the present invention,but, for example, when a vector of the present invention prepared basedon pTrcHis is used, it is preferable to select Escherichia coli strain.

The host can be transformed by the vector of the present invention inthe usual way. For example, the host can be transformed by introducingthe vector of the present invention into the host by a method using acommercially available transfection reagent, a DEAE-dextran method,electroporation or the like.

The transformant of the present invention obtained in this manner can beused in the enzyme production process of the present invention describedbelow.

<5> Enzyme Production Process of the Present Invention

The enzyme production process of the present invention is a process forproducing a chondroitin polymerase, comprising growing the transformantof the present invention; and collecting a chondroitin polymerase fromthe grown material.

The term “growing” as used herein means a general idea which includesgrowth of cells or microorganism as the transformant of the presentinvention itself and growth of an animal, insect or the like into whichcells as the transformant of the present invention are incorporated.Also, the term “grown material” as used herein means a concept whichincludes a medium (supernatant of culture medium) and cultured hostcells after growth of the transformant of the present invention,secreted matter, excreted matter and the like.

Growth conditions (medium, culture condition and the like) areappropriately selected based on the host to be used.

According to the enzyme production process of the present invention,various forms of chondroitin polymerase can be produced based on thetransformant to be used.

For example, when a transformant transformed with an expression vectorconstructed for expressing a fusion protein with a marker peptide isgrown, a chondroitin polymerase fused with the marker peptide isproduced. Specifically, for example, a chondroitin polymerase fused withHis tag is produced by growing a transformant transformed with anexpression vector constructed for effecting expression of a protein inwhich the amino acid sequence represented by SEQ ID NO:12 iscontinuously fused to a position just before the amino acid sequencerepresented by SEQ ID NO: 2. Particularly, it is preferable to use atransformant transformed with an expression vector constructed byconnecting the nucleotide sequence represented by SEQ ID NO: 11continuously to a position just before the nucleotide sequencerepresented by SEQ ID NO: 1.

The chondroitin polymerase can be collected from the grown matter byknown protein extraction and purification methods based on the form ofthe produced chondroitin polymerase.

For example, when the chondroitin polymerase is produced in a solubleform secreted into a medium (supernatant of culture medium), the mediummay be collected and used directly as the chondroitin polymerase. Also,when the chondroitin polymerase is produced in a soluble form secretedinto the cytoplasm or in an insoluble form (membrane binding), thechondroitin polymerase can be extracted by cell disintegration such as amethod using a nitrogen cavitation apparatus, homogenization, glassbeads mill, sonication, an osmotic shock method, freezing-thawing, etc.,surfactant extraction, a combination thereof or the like, and theextract may be used directly as the chondroitin polymerase.

The chondroitin polymerase can be further purified from the media orextracts, which is preferable. The purification may be either imperfectpurification (partial purification) or perfect purification, which canbe appropriately selected based on, e.g., the object using thechondroitin polymerase, and the like.

Examples of the purification method include salting out by ammoniumsulfate, sodium sulfate, etc., centrifugation, dialysis,ultrafiltration, adsorption chromatography, ion exchange chromatography,hydrophobic chromatography, reverse phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, a combination thereof and the like.

Production of the chondroitin polymerase can be confirmed by analyzingits amino acid sequence, actions, substrate specificity and the like.

<6> Synthesizing Agent of the Present Invention

The synthesizing agent of the present invention is a sugar chainsynthesizing agent, comprising an enzyme protein which comprises anamino acid sequence represented by the following (A) or (B) and hasenzymic activities of the following (i) and (ii):

(A) the amino acid sequence represented by SEQ ID NO: 2;(B) an amino acid sequence in which one or a few two amino acid residue(s) in the amino acid sequence represented by SEQ ID NO:2 are deleted,substituted, inserted or transposed;(i) GlcUA and GalNAc are alternately transferred to a non-reducedterminal of a sugar chain from a GlcUA donor and a GalNAc donor,respectively;(ii) GlcNAc is transferred to a non-reduced terminal of a sugar chainhaving GluUA on the non-reduced terminal from a GlcNAc donor.

As the “enzyme protein which comprises an amino acid sequencerepresented by the following (A) or (B) and has enzymic activities ofthe following (i) and (ii)” which is the active ingredient of thesynthesizing agent of the present invention, the enzyme of the presentinvention or the protein of the present invention can be used as such.

The synthesizing agent of the present invention is a result of applyingthe “GalNAc transferring action”, “GlcNAc transferring action” and“GlcUA transferring action” of the enzyme of the present invention andprotein of the present invention as a sugar chain synthesizing agent.

The synthesizing agent of the present invention is used for thesynthesis of sugar chains. The term “synthesis of sugar chain” or “sugarchain synthesis” as used herein means a concept including elongation ofa certain sugar chain by transferring and adding a monosaccharide to thesugar chain. For example, a concept of transferring and adding amonosaccharide such as GlcUA, GalNAc, GlcNAc or the like to a sugarchain such as chondroitin, chondroitin sulfate, hyaluronic acid and thelike to elongate the sugar chain is included in the term “sugar chainsynthesis” as used herein.

The form of the synthesizing agent of the present invention is notlimited, and it may be any one of a solution form, a frozen form, afreeze-dried form and an immobilized enzyme form in which it is linkedto a carrier. Also, it may contain other components (e.g.,pharmaceutically acceptable carrier, carrier which is acceptable forreagent, etc.), so long as they do not have influence on the activity ofchondroitin polymerase.

<7> Sugar Chain Production Process of the Present Invention

The sugar chain production process of the present invention uses thesynthesizing agent of the present invention and is divided into thefollowing five methods in response to the sugar donors and acceptorsubstrates.

(1) Sugar Chain Production Process of the Present Invention 1

A process for producing a sugar chain represented by the followingformula (3), which comprises at least a step of allowing thesynthesizing agent of the present invention to contact with a GalNAcdonor and a sugar chain represented by the following formula (1):

GlcUA-X—R¹  (1)

GalNAc-GlcUA-X—R¹  (3)

(2) Sugar Chain Production Process of the Present Invention 2

A process for producing a sugar chain represented by the followingformula (4), which comprises at least a step of allowing thesynthesizing agent of the present invention to contact with a GlcNAcdonor and a sugar chain represented by the following formula (1):

GlcUA-X—R¹  (1)

GlcNAc-GlcUA-X—R¹  (4)

(3) Sugar Chain Production Process of the Present Invention 3

A process for producing a sugar chain represented by the followingformula (5), which comprises at least a step of allowing thesynthesizing agent of the present invention to contact with a GlcUAdonor and a sugar chain represented by the following formula (2):

GalNAc-GlcUA-R²  (2)

GlcUA-GalNAc-GlcUA-R²  (5)

(4) Sugar Chain Production Process of the Present Invention 4

A process for producing a sugar chain selected from the followingformulae (6) and (8), which comprises at least a step of allowing thesynthesizing agent of the present invention to contact with a GalNAcdonor and a GlcUA donor and a sugar chain represented by the followingformula (1):

GlcUA-X—R¹  (1)

(GlcUA-GalNAc)n-GlcUA-X—R¹  (6)

GalNAc-(GlcUA-GalNAc)n-GlcUA-X—R¹  (8)

(5) Sugar Chain Production Process of the Present Invention 5

A process for producing a sugar chain selected from the followingformulae (7) and (9), which comprises at least a step of allowing thesynthesizing agent of the present invention to contact with a GalNAcdonor, a GlcUA donor and a sugar chain represented by the followingformula (2):

GalNAc-GlcUA-R²  (2)

(GalNAc-GlcUA)n-GalNAc-GlcUA-R²  (7)

GlcUA-(GalNAc-GlcUA)n-GalNAc-GlcUA-R²  (9)

In each of the formulae, X represents GalNAc or GlcNAc, and R¹ and R²each represents any group. R¹ and R² are the same or different from eachother.

Examples of R¹ and R² include a sugar chain having a chondroitinbackbone, a sugar chain having a hyaluronic acid backbone and the like.

The sugar chain represented by formula (1) is preferably chondroitinsulfate (particularly chondroitin sulfate C), chondroitin or hyaluronicacid having GlcUA on its non-reduced terminal, or an oligosaccharidethereof.

The sugar chain represented by formula (2) is preferably chondroitinsulfate (particularly chondroitin sulfate C) or chondroitin havingGalNAc on its non-reduced terminal, or an oligosaccharide thereof.

As the GalNAc donor, nucleoside diphosphate-GalNAc is preferable, andUDP-GalNAc is particularly preferable. Furthermore, as the GlcNAc donor,nucleoside diphosphate-GlcNAc is preferable, and UDP-GlcNAc isparticularly preferable. Moreover, as the GlcUA donor, nucleosidediphosphate-GlcUA is preferable, and UDP-GlcUA is particularlypreferable.

The method for carrying out “contact” is not particularly limited, solong as the enzyme reaction is generated by mutual contact of moleculesof the enzyme of the present invention (or the protein of the presentinvention) contained in the synthesizing agent of the present invention,a donor and an acceptor (sugar chain). For example, the contact may becarried out in a solution in which the three components are dissolved.Also, the enzyme reaction can be carried out continuously using animmobilized enzyme in which chondroitin polymerase contained in thesynthesizing agent of the present invention is linked to an appropriatesolid phase (beads or the like) or a membrane type reactor usingultrafiltration membrane, dialysis membrane or the like. Also, theenzyme reaction can be carried out by linking the acceptor to a solidphase similar to the method described in WO 00/27437. In addition, abioreactor which regenerate (synthesize) the donor may be used incombination.

In addition, in the above processes (4) and (5), it is not alwaysnecessary to contact the GalNAc donor and GlcUA donor simultaneouslywith the synthesizing agent of the present invention and the sugar chainrepresented by formula (1) or (2), and the donors may be allowed tocontact alternately.

The conditions for carrying out the enzyme reaction is not particularlylimited, so long as they are conditions under which the enzyme of thepresent invention (or the protein of the present invention) canfunction, but it is preferable to carry out the reaction at aroundneutral pH (e.g., about pH 7.0 to 7.5), and it is more preferable tocarry out the reaction in a buffer having the buffering action under thepH. Also, the temperature in this case is not particularly limited, solong as the activity of the enzyme of the present invention (or theprotein of the present invention) is retained, but a temperature ofapproximately from 25° C. to 30° C. can be exemplified. Also, when thereis a substance which increases the activity of the enzyme of the presentinvention (or the protein of the present invention), the substance maybe added. For example, it is preferable to allow Mn²⁺ and the like tocoexist. The reaction time can be determined optionally by those skilledin the art in response to the amounts of the synthesizing agent of thepresent invention, donor and acceptor to be used and other reactionconditions.

Isolation and the like of sugar chain from the formed product can becarried out by known methods.

Also, a sulfated saccharide such as chondroitin sulfate or the like canbe produced by using the synthesizing agent of the present invention(chondroitin polymerase) and a sulfotransferase in combination.

For example, a sulfated saccharide such as chondroitin sulfate or thelike can be produced by simultaneously carrying out formation ofchondroitin and transfer of sulfate group in the above sugar chainproduction process, by further allowing a sulfate group donor(3′-phosphoadenosine 5′-phosphosulfate (PAPS) or the like) and asulfotransferase to coexist. The sulfotransferase may be used as animmobilized enzyme by linking it to an appropriate solid phase (beads orthe like) similar to the above case or allowed to react continuouslyusing a membrane type reactor using ultrafiltration membrane, dialysismembrane or the like. In this case, a bioreactor which regenerate(synthesize) the sulfate group donor may be used in combination.

The sulfotransferase which can be used herein may be any enzyme whichcan transfer a sulfate group to chondroitin and can be appropriatelyselected from known enzymes based on the kind of desired chondroitinsulfate. Also, two or more kinds of sulfotransferase having differentsulfate group transferring positions may be used in combination.

Chondroitin 6-O-sulfotransferase (J. Biol. Chem., 275 (28), 21075-21080(2000)) can be exemplified as the sulfotransferase. However, there is nolimitation thereto and other enzymes can also be used.

<8> Probe of the Present Invention

The probe of the present invention is a hybridization probe comprising anucleotide sequence complementary to the nucleotide sequence representedby SEQ ID NO:1 or a part thereof.

The probe of the present invention can be obtained by preparing anoligonucleotide comprising a nucleotide sequence complementary to thenucleotide sequence represented by SEQ ID NO:1 or a part thereof, andlabeling it with a label suitable for hybridization (e.g.,radioisotope).

The size of the oligonucleotide is appropriately selected based onconditions and the like of the hybridization using the probe of thepresent invention.

It is expected that the probe of the present invention becomes a usefultool for examining biological functions of chondroitin sulfate. This isbecause chondroitin sulfate is broadly expressed and plays an importantrole in a large number of tissues, particularly in the brain. It isconsidered that the probe is also useful in searching for therelationship between genes and diseases.

<9> Catalyst of the Present Invention

The catalyst of the present invention is a glycosyltransfer catalystwhich comprises an enzyme protein comprising an amino acid sequenceselected from the following (A) and (B), and is capable of transferringGlcUA, GalNAc and GlcNAc from a GlcUA donor, a GalNAc donor and a GlcNAcdonor, respectively to a non-reduced terminal of a sugar chain:

(A) the amino acid sequence represented by SEQ ID NO:2;(B) an amino acid sequence in which one or a few amino acid residue(s)in the amino acid sequence represented by SEQ ID NO: 2 are deleted,substituted, inserted or transposed.

As the “enzyme protein comprising an amino acid sequence represented by(A) or (B)” which is the active ingredient of the catalyst of thepresent invention, the enzyme of the present invention or protein of thepresent invention can be used as such.

The catalyst of the present invention is a result of applying the“GalNAc transferring action”, “GlcNAc transferring action” and “GlcUAtransferring action” of the enzyme of the present invention and proteinof the present invention as a glycosyltransfer catalyst.

The catalyst of the present invention can be used for transfer of GlcUA,GalNAc or GlcNAc. For example, it can be used for transferring amonosaccharide such as GlcUA, GalNAc, GlcNAc or the like to anon-reduced terminal of a sugar chain such as chondroitin, chondroitinsulfate, hyaluronic acid or the like.

The form of the catalyst of the present invention is not limited, and itmay be any one of a solution form, a frozen form, a freeze-dried formand an immobilized enzyme form in which it is linked to a carrier. Also,it may contain other components (e.g., pharmaceutically acceptablecarrier, carrier which is acceptable for reagent, etc.), so long as theydo not have influence on the transferring activity of GlcUA, GalNAc orGlcNAc.

Since the enzyme of the present invention and the protein of the presentinvention can transfer GlcUA and GalNAc alternately as a singlemolecule, they are markedly useful as tools for the large scaleproduction of sugar chain having a chondroitin backbone (chondroitin,chondroitin sulfate and the like), as the active ingredients of thesynthesizing agent of the present invention and the catalyst of thepresent invention, reagents or the like. Also, the DNA of the presentinvention is markedly useful as a tool for the large scale production ofsuch enzyme of the present invention and protein of the presentinvention. The vector of the present invention is markedly useful,because it can retain the DNA of the present invention stably andfunction effectively and efficiently. The transformant of the presentinvention is also markedly useful, because not only it can retain thevector of the present invention stably and function effectively andefficiently, but also it can be used as such for the large scaleproduction of the enzyme of the present invention and the protein of thepresent invention. In addition, the enzyme production process of thepresent invention is markedly useful for the large scale production ofthe enzyme of the present invention and protein of the presentinvention. Also, the sugar chain production process of the presentinvention is markedly useful for the large scale production of sugarchain having the chondroitin backbone (chondroitin, chondroitin sulfateand the like). The synthesizing agent of the present invention and thecatalyst of the present invention are markedly useful, because they canbe used in the sugar chain production process of the present invention.

Since high quality and uniform chondroitin polymerase can be producedconveniently, quickly and in a large scale by the present invention, lowcost products can be provided for the industrial field and therefore thepresent invention has markedly high availability.

The present invention is explained in detail based on Examples. However,the present invention is not limited thereto.

UDP-[¹⁴C]GlcUA, UDP-[³H]GalNAc and UDP-[¹⁴C]GlcNAc used in Examples werepurchased from NEN Life Sciences. Also, UDP-GlcUA, UDP-GalNAc andUDP-GlcNAc were purchased from Sigma.

Example 1 Cloning of Chondroitin Polymerase Gene (1) Preparation of DNALibrary

Escherichia coli strain K4 (serotype O5:K4 (L):H4, ATCC 23502) wascultured at 37° C. overnight in 50 ml of LB medium. The cells werecollected by centrifugation (3,800 rpm, 15 minutes), suspended in 9 mlof 10 mM Tris-HCl (pH 8.0) buffer containing 1 mMethylenediaminetetraacetic acid (EDTA) (hereinafter referred to as “TE”)and then treated at 37° C. for 1 hour by adding 0.5 ml of 10% SDS and 50μl of proteinase K (20 mg/ml, Boehringer Mannheim). To the suspension,10 ml of PCI solution (phenol:chloroform:isoamyl alcohol=25:24 : 1) wasadded, followed by stirring for 30 minutes, and the resulting mixturewas centrifuged (3,800 rpm, 15 minutes) to collect the water layer andthe intermediate layer insoluble matter and again centrifuged (10,000rpm, 30 minutes). The supernatant was recovered and 50 μl of RNase A (20mg/ml, Sigma) was added thereto for reaction at 37° C. for 1 hour. Tothe treated solution, 10 ml of PCI solution was added, followed bystirring for 30 minutes, and the resulting mixture was centrifuged(3,800 rpm, 15 minutes) to collect the water layer and again centrifuged(10,000 rpm, 30 minutes). The supernatant was recovered and dialyzedagainst 2,000 ml of TE at 4° C. overnight, and the thus dialyzedsolution (7.5 ml) was used as a chromosomal DNA solution (DNAconcentration, 0.9 mg/ml). The thus obtained K4 strain-derivedchromosomal DNA solution (120 μl) was digested using a restrictionenzyme Sau3 A1 (4 units: NEB) at 37° C. for 30 minutes and thensubjected to 0.3% agarose gel electrophoresis, and then the agarose gelcorresponding to the DNA of about 7 to 11 kbp was cut out. The gel thuscut out was put into a 1.5 ml capacity tube having a hole on its bottompricked with a needle and, together with the tube, inserted into a 2 mlcapacity tube and centrifuged (8,000 rpm, 1 minute) to break up the gel.Neutralized phenol in an almost the same volume of the gel was addedthereto, followed by vigorously stirring and then the resulting mixturewas frozen at −80° C. Thirty minutes thereafter, the temperature wasreturned to room temperature to melt the mixture, followed bycentrifugation (13,000 rpm, 5 minutes). The resulting aqueous layer wascollected, the same volume of PCI solution was added thereto, followedby stirring, and then the resulting mixture was centrifuged (13,000 rpm,5 minutes). The aqueous layer was collected, 1/10 volume of 3 M sodiumacetate solution and the same volume of 2-propanol were added thereto toprecipitate DNA, and the precipitate was then collected bycentrifugation (13,000 rpm, 30 minutes). To the thus collectedprecipitate, 70% ethanol solution was added, followed by centrifugation(13,000 rpm, 5 minutes), and then the resulting precipitate wasdissolved by adding 100 μl of TE. In order to concentrate the resultingsolution, DNA was precipitated by adding 10 μl of 3 M sodium acetatesolution and 300 μl of ethanol and then recovered by centrifugation(13,000 rpm, 20 minutes). To the thus collected precipitate, 70% ethanolsolution was added, followed by centrifugation (13,000 rpm, 5 minutes),and the resulting precipitate was dissolved in 4 μl of purified water toobtain a chromosomal DNA fragment solution. The DNA fragment solution (2μl) was inserted into a λ phage vector (λ EMBL3: STRATAGENE) which hadbeen treated with restriction enzymes (BamHI (80 units: NEB) and EcoRI(80 units: NEB)) and subjected to packaging using a packaging kit(Gigapack III Gold Packaging Extract, STRATAGENE) in accordance with themanufacture's instructions, and then Escherichia coli strain (NM539) wasinfected with the λ phage and propagated to prepare a K4 chromosomal DNAlibrary.

(2) Preparation of Probe

Among 3 regions of the K antigen gene cluster moiety of Escherichia colistrain K5 (serotype 010:K5 (L):H4, ATCC 23506) having known sequences(Mol. Microbiol., 17 (4), 611-620 (1995)), while interposing the Kantigen polysaccharide-specific region R-II (gene bank accession NO.X77617), a primer set (CS-S 5′-ACCCAACACTGCTACAACCTATATCGG-3′ (SEQ IDNO:5); CS-AS 5′-GCGTCTTCACCAATAAATACCCACGAAACT-3′ (SEQ ID NO: 6)) toobtain a DNA fragment of about 1 kbp from the 3′-terminal of the R-Iregion (gene bank accession NO. X74567), and another primer set (TM-S5′-CGAGAAATACGAACACGCTTTGGTAA-3′ (SEQ ID NO:7); TM-AS5′-ACTCAATTTTCTCTTTCAGCTCTTCTTG-3′ (SEQ ID NO:8)) to obtain a DNAfragment of about 1 kbp from the 5′-terminal of the R-III region (genebank accession NO. X53819) were selected and prepared.

Using the respective primer sets for R-I and R-III and using, as thetemplate, genome DNA fragments of the strain K4 extracted and purifiedafter Sau3 A1 treatment and subsequent agarose gel electrophoresis inthe above (1), polymerase chain reaction (PCR) (94° C., 1 min-(94° C.,45 sec-47° C., 30 sec-72° C., 5 min) 30 cycles-72° C., 10 min (for R-I),94° C., 1 min-(94° C., 45 sec-50° C., 30 sec-72° C., 5 min) 30cycles-72° C., 10 min (for R-III)) was carried out to obtain K4-derivedR-I region 1.3 kbp (K4 RI3) and R-III region 1.0 kbp (K4 RIII5) DNAfragments. Nucleotide sequences of the thus obtained DNA fragments weredetermined using ABI PRISM 310 Genetic Analyzer (Perkin-Elmer). Thehomology with the strain K5 DNA at the same genetic positions was 96%and 95%, respectively.

(3) Gene Cloning of K4 R-II Region

Using respective R-I region and R-III region DNA fragments (K4 RI3 andK4 RIII5) as probes, K4 antigen gene clusters were screened from the K4chromosomal DNA library obtained in the above (1). Escherichia coli(strain NM539) culture (30 μl) was infected with the K4 chromosomal DNAlibrary (λ phage 40 μl) (37° C., 15 minutes), 10 ml of top agarose wasadded thereto, the resulting mixture was spread on LB plate mediumcontained in five 10×14 cm Petri dishes, followed by culturing at 37° C.for 9.5 hours to form plaques. Two 9×13 cm membranes (Hybond-N+:Amersham Pharmacia Biotech) were prepared for each plate, and the firstand second membranes were put on the medium for 1 minute and 3 minutes,respectively. After removing excess moisture, each membrane was soakedfor 2 minutes in 0.5 M NaOH solution containing 1.5 M NaCl to carry outdenaturation treatment and then soaked for 3 minutes in 1 M Tris-HCl (pH7.4) containing 1.5 M NaCl to carry out neutralization treatment. Afterdrying, the membrane was baked at 80° C. for 2 hours to prepare afilter. The filter was subjected to pre-hybridization treatment at 65°C. for 1 hour, hybridized with [³² P]-labeled K4 RI3 at 64° C. overnight(15 hours) in 0.5 M Church phosphate buffer (pH 7.2), 1 mM EDTA and 7%SDS, and then washed three times with 40 mM Church phosphate buffer (pH7.2) containing 1% SDS (65° C., each for 15 minutes). The filter wasdried and then exposed to an X-ray film to thereby obtain 30 positiveplaques. The presence of K4 RI3 was confirmed for each of them by PCR,and 7 plaques among them were subjected to the second screening. Next,the filter hybridized with K4 RI3 was boiled in 0.5% SDS solution for 3minutes to remove K4 RI3 and then dried to be used as a K4 RIII5hybridization filter. The filter was subjected to pre-hybridizationtreatment at 65° C. for 1 hour, hybridized with [³² P]-labeled K4 RIII5at 64° C. overnight and then washed three times with 40 mM Churchphosphate buffer containing 1% SDS. The filter was dried and thenexposed to an X-ray film to thereby obtain 29 positive plaques. Thepresence of K4 RIII5 was confirmed for each of them by PCR, and 18plaques among them were subjected to the second screening.

In the second screening, LB plate medium of φ 9 cm was used, andpositive plaques were obtained by the same method of first screening.

After the first and second screening, 4λ phage clones were obtained fromthe R-I region, and 10 clones from the R-III region. Each of the cloneswas subjected to enzyme treatment with EcoRI (10 units: NEB), SalI (10units: NEB) and BamHI (10 units: NEB), each independently orsimultaneously in various combinations, and their restriction maps wereprepared based on the size of fragments observed by electrophoresis(FIG. 1).

Among these clones, one clone (CS23, insertion region 15.4 kbp) is a DNAclone prepared based on the R-III region probe, but since it also showeda weak reaction with the R-I region probe, 5′-terminal sequence of theinsertion region was examined to find a sequence completely coincidedwith the 3′-end of the R-I region probe. Since both of the DNA fragmentsof the R-I region and R-III region were contained in the insertionregion, the clone was judged as a clone which contains all of the R-IIregion of the K antigen gene cluster of the strain K4 .

(4) Genetic Analysis of K4 R-II Region

Subcloning of the above CS23 clone was carried out to carry out itssequencing. First, each of about 3 kbp and 8 kbp DNA fragments obtainedby treating the CS23 clone with EcoRI and 2 kbp, 5 kbp and 7 kbp DNAfragments obtained by treating it with Sail was ligated with a cloningvector (pBluescript II KS(−)) and integrated into Escherichia colistrain (XLI-Blue) to obtain a clone having different direction ofinsert. By repeating “treatment of multi-cloning sites of the vectorwith various restriction enzymes-ligation-transformation” on the clone,22 plasmids having partial DNA fragments of the R-II region wereobtained. By carrying out sequencing of the insertion DNA fragments andconnecting them, complete gene sequence of the K4 R-II region wasdetermined (SEQ ID NO: 3). SEQ ID NO: 4 depicts the deduced amino acidsequence encoded by polynucleotides 3787 to 5847 of SEQ ID NO: 3.

(5) Identification of Chondroitin Polymerase Gene

As a result of the analysis of the K4 strain R-II region DNA sequence,the presence of 8 open reading frames (ORF) was predicted (FIG. 2).

Among them, the third position ORF counting from the R-III side (2,061bp (nucleotide numbers 3,787 to 5,847 in SEQ ID NO: 3, sequence of 2,058bp by excluding the termination codon is shown in SEQ ID NO:1), 686 asthe number of amino acids, molecular weight obtained by calculation79,256 (SEQ ID NO:2)) showed 59% of homology with a Pasteurellamultocida hyaluronic acid synthase (class 2 type pmHAS; J. Biol. Chem.,273 (14), 8454-8458 (1998)). Also, the first position ORF counting fromthe R-III side (1,017 bp (nucleotide numbers 643 to 1,659 in SEQ IDNO:3), 339 as the number of amino acids) showed 60% of homology withPasteurella multocida UDP-glucose-4-epimerase (Submitted (29 Oct. 1996)Genetics and Microbiology, Autonomus University of Barcelona, Edifici C,Bellaterra, BCN 08193, Spain), the fourth position ORF (1,332 bp(nucleotide numbers 5,877 to 7,207 in SEQ ID NO:3)) showed high homology(98%) with Insertion Sequence 2 (Nucleic Acids Res., 6(3), 1111-1122(1979)), and the seventh position ORF (1,167 bp (nucleotide numbers11,453 to 12,619 in SEQ ID NO:3), 389 as the number of amino acids)showed 65% of homology with the kfiD (Mol. Microbiol., 17(4), 611-620(1995)) gene (encodes UDP-glucose dehydrogenase) of Escherichia colistrain (K5). Also, since the eighth position ORF (1,035 bp (nucleotidenumbers 13,054 to 14,088 in SEQ ID NO:3), 345 as the number of aminoacids) contained a DXD motif common to glycosyltransferase, it wasconsidered that it has a sugar transferring activity. Regarding theremaining three ORFs (Nos. 2, 5 and 6 (nucleotide numbers 1,849 to3,486, 7,210 to 8,673 and 9,066 to 10,631, respectively, in SEQ IDNO:3), no homology was found.

Example 2 Expression and Enzyme Activity of Chondroitin PolymeraseProtein

(1) In order to confirm that the K4 R-II region ORF (No. 3) is achondroitin polymerase gene, primers having restriction enzyme cut sitesand interposing the corresponding ORF moiety (K4C-SP5′-CGGGATCCCGATGAGTATTCTTAATCAAGC-3′ (SEQ ID NO:9); K4C-AS5′-GGAATTCCGGCCAGTCTACATGTTTATCAC-3′ (SEQ ID NO:10)) were prepared andPCR (94° C., 1 min-(94° C., 30 sec-59° C., 30 sec-74° C., 3 min) 20cycles-74° C., 10 min) was carried out. The PCR product was subjected to0.7% agarose gel electrophoresis and extracted and purified using a gelextraction kit (QIAGEN). After treating with restriction enzymes (BamHIand EcoRI), the product was again subjected to 0.7% agarose gelelectrophoresis and extracted and purified in the same manner to be usedas an insert.

The insert prepared in the above was inserted into an expression vector(pTrcHisC: Invitrogen; containing the nucleotide sequence represented bySEQ ID NO:4) which had been treated with restriction enzymes (BamHI andEcoRI) and CIP, at 16° C. spending 1 hour in the presence of T4 DNAligase, and transformed into Escherichia coli strain (TOP10). Byculturing the Escherichia coli strain (LB plate medium containingampicillin, 37° C., overnight), 7 colonies were obtained. One clonecontaining a plasmid into which the above insert was correctly insertedwas selected from them. The Escherichia coli was cultured (37° C.,overnight) in 1.5 ml of LB medium containing ampicillin (100 μg/ml), and50 μl of the cultured cell suspension was inoculated into 50 ml of LBmedium containing ampicillin (100 μg/ml) and cultured at 37° C. untilOD₆₀₀ became 0.6. To the culture, 1 ml of 0.5 M isopropyl1-thio-β-D-galactoside (IPTG) was added (final concentration: 1 mM) andinduction was carried out at 37° C. for 3 hours. The cells werecollected by centrifugation (10,000 rpm, 30 minutes) and suspended byadding 4 ml of a lysis buffer (50 mM NaH₂ PO₄ (pH 8.0) containing 300 mMNaCl and 10 mM imidazole). To the suspension, 4 mg of lysozyme (Sigma)was added, the resulting mixture was allowed to stand on ice for 30minutes, and then the cells were disrupted by three times ofultrasonication, each for 10 seconds, using a sonicator. The supernatantwas collected by centrifugation (10,000 rpm, 30 minutes) and applied toNi-NTA agarose column (carrier 1 ml, equilibrated with the lysis buffer;QIAGEN), followed by stirring using a rotor (4° C. for 1 hour). Thecarrier was sunk by setting up the column and then the column was washedtwice using 4 ml for each of a wash buffer (50 mM NaH₂ PO₄ (pH 8.0)containing 300 mM NaCl and 20 mM imidazole). Next, proteins were elutedby passing 4 times 0.5 ml for each of an elution buffer (50 mM NaH₂ PO₄(pH 8.0) containing 300 mM NaCl and 250 mM imidazole). The eluatecontaining the protein of interest (1 ml) was dialyzed at 4° C. for 2days against 500 ml of PBS (phosphate buffered saline) containing 20%glycerol to thereby obtain about 0.5 ml (protein content 0.4 mg/ml) ofdialyzed solution (solution of the enzyme of the present invention(protein of the present invention)).

Western blotting of the thus obtained protein was carried out by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In theSDA-PAGE, 10% gel was used. Protein was detected by Coomassie BrilliantBlue staining. The Western blotting was carried out by transferringprotein in the SDS-PAGE gel onto a nitrocellulose membrane, blocking themembrane with 5% skimmed milk (dissolved in 25 mM Tris-HCl (pH 7.5)containing 150 mM NaCl and 0.05% Tween 20 (this solution is to be calledTBS-T)) and then treating it with anti-tetra-His antibody (Qiagen).After washing several times with TBS-T, this membrane was treated withperoxidase-linked anti-mouse IgG. After washing with TBS-T, the reactedprotein was detected by ECL detection system (Amersham).

As a result, the protein showed a band at around 80 kDa by the Westernblotting analysis using SDS-PAGE and anti-tetra-His antibody. On theother hand, an immunologically reacting band was not detected in theculture extract of a control (expression vector having no insert).

(2) Analysis of Enzyme Activity (Analysis of GalNAc Transfer Activity)

The enzyme of the present invention (2 μg), hexasaccharide of sharkcartilage chondroitin sulfate C, purified by degrading with testicularhyaluronidase, as the acceptor (70 pmol) and UDP-GalNAc (3 nmol),UDP-GlcUA (3 nmol) and UDP-[³H]GalNAc (0.1 nmol, 0.1 μCi) as the donorswere added to 50 mM Tris-HCl (pH 7.2) containing 20 mM MnCl₂, 0.1 M(NH₄)₂ SO₄ and 1 M ethylene glycol, and the total volume was adjusted to50 μl, and then the reaction was carried out at 30° C. for 30 minutesand the enzyme was heat-inactivated. To the reaction solution, 3 volumesof 95% ethanol containing 1.3% potassium acetate was added, and thesample was centrifuged at 10,000×g for 20 minutes. The precipitate wasdissolved in 50 μl of distilled water and applied to a Superdex Peptidecolumn (300×φ10 mm: Amersham Biosciences, chromatography conditions;buffer: 0.2 M NaCl, flow rate: 0.5 ml/rain), and the eluate wasfractionated at 0.5 ml and the radioactivity (count of [³H]) of eachfraction was measured using a scintillation counter. Chondroitinsynthesizing activity was determined by calculating the amount ofradioactivity incorporated into fractions of higher molecular weightthan the acceptor substrate. The results are shown in FIG. 3. In FIG. 3,the closed squares indicate radioactivity when hexasaccharide ofchondroitin sulfate C was used as the acceptor, open triangles indicateradioactivity when the enzyme reaction product was treated withchondroitinase ABC and closed circles indicates control (whereinheat-inactivated enzyme of the present invention was used).

As a result, elution position of the radioactivity appeared at a highermolecular weight side than the hexasaccharide of chondroitin sulfate C(the broad peak having its top at around 5,000 Da) (closed squares inFIG. 3). Also, when the enzyme reaction product was treated withchondroitinase ABC, the high molecular weight peak was shifted to aposition corresponding to the disaccharide fraction (open triangles inFIG. 3). When disaccharide composition of this chondroitinase ABC digestwas analyzed using a high performance liquid chromatography (HPLC), onlyan un-sulfated, unsaturated chondroitin disaccharide (ΔdiOS) wasdetected.

Also, the enzyme reaction product was completely digested bychondroitinase ACII treatment too, but not digested by Streptomyceshyaluronidase and heparitinase I.

Based on the above, it was shown that the thus obtained enzyme of thepresent invention at least transfers GalNAc to the hexasaccharide ofchondroitin sulfate C from UDP-GalNAc (donor). This specific activitywas 3.25±0.64 nmol GalNAc/min/mg protein.

(3) Analysis of Size of Enzyme Reaction Product

The size of the enzyme reaction product was examined by carrying out theenzyme reaction and chromatography in the same manner as in the above“(2) Analysis of enzyme activity”. The results are shown in FIG. 4.

It was confirmed from FIG. 4 that the enzyme of the present inventionobtained in the above at least transfers GalNAc to an acceptor (anoligosaccharide having the chondroitin backbone (hexasaccharide ofchondroitin sulfate C prepared using testicular hyaluronidase)) from aGalNAc donor (UDP-GalNAc) to thereby form chondroitin having a molecularweight of 10,000 to 20,000 or more.

(4) Analysis of Specificity of Donor Substrate

Using UDP-[¹⁴C]GlcUA, UDP-[¹⁴C]GlcNAc or UDP-[³H]GalNAc as the donor,transferring activity to the following acceptors was examined inaccordance with the method described in the above “(2) Enzyme activitymeasurement”. Products after the enzyme reaction were analyzed by gelfiltration.

Results of the use of hexasaccharide or heptasaccharide of chondroitinsulfate C as the acceptor are shown in FIG. 5. Also, the closed circlein FIG. 5 indicates a control (a case in which heat-inactivated enzymeof the present invention was used).

(A) Hexasaccharide of chondroitin sulfate C (A product purified bydegrading shark cartilage chondroitin sulfate C with testicularhyaluronidase, and the non-reduced terminal is GlcUA)

Heptasaccharide alone was synthesized when UDP-GalNAc alone was used asthe donor (closed lozenge in FIG. 5).

Substantial transfer was not found when UDP-GlcUA alone was used as thedonor (closed triangle in FIG. 5).

Although very little, transfer of GlcNAc was found when UDP-GlcNAc alonewas used as the donor. This transferring activity was 6.3% based on 100%activity in the case of the use of UDP-GalNAc as the donor (closedsquare in FIG. 5). However, polysaccharides having a size ofoctasaccharide or more were not obtained even when both of UDP-GlcNAcand UDP-GlcUA were used together with this.

In addition, since incorporation of the radioactivity was not found inthe absence of acceptor substrate, it was suggested that an acceptorsubstrate is essential for the synthesis of chondroitin by this enzyme.

(B) Heptasaccharide of chondroitin sulfate C (A product obtained byallowing the enzyme of the present invention to react with the abovehexasaccharide of chondroitin sulfate C and thereby linking one residueof GalNAc to the non-reduced terminal of the hexasaccharide)

Octasaccharide alone was synthesized when UDP-GlcNAc alone was used asthe donor (open triangle in FIG. 5).

When either UDP-GalNAc or UDP-GlcNAc alone was used as the donor,substantial transfer was not found in each case (respectively, openlozenge and open square in FIG. 5).

Also, results of carrying out similar tests each independently are shownin Table 1. Also, the term “CS” in Table 1 means chondroitin sulfate C.Also, parenthesis in the table shows length of sugar chain after theenzyme reaction and “-” means that the labeled UDP-sugar was nottransferred to the corresponding acceptor substrate.

TABLE 1 Chondroitin polymerase-specific activity (nmol/min/mg protein)Donor substrate Acceptor substrate Labeled Unlabeled CS CS UDP-sugarUDP-sugar hexasaccharide heptasaccharide UDP-[³H]GalNAc none 0.59 ± 0.16(hepta)  0.0 ± 0.0 (—) UDP-[¹⁴C]GlcNAc none 0.04 ± 0.02 (hepta)  0.0 ±0.0 (—) UDP-[¹⁴C]GlcUA none  0.0 ± 0.0 (—) 0.53 ± 0.08 (octa)UDP-[³H]GalNAc UDP-GlcUA 3.25 ± 0.64 (poly) not measured UDP-[¹⁴C]GlcUAUDP- 2.75 ± 0.28 (poly) not measured GalNAc UDP-[¹⁴C]GlcNAc UDP-GlcUA0.05 ± 0.02 (poly) not measured UDP-[¹⁴C]GlcUA UDP-  0.0 ± 0.0 (—) notmeasured GlcNAc

Prom the above results, it was shown that the enzyme of the presentinvention obtained in the above transfers GalNAc from UDP-GalNAc to asugar chain (acceptor) having a chondroitin backbone whose non-reducedterminal is GlcUA. Also, it was shown that when the acceptor is used,the enzyme of the present invention obtained in the above shows theactivity to transfer GlcNAc from UDP-GlcNAc, but the activity is farlower than its GalNAc transferring activity. Also, it was shown thatwhen the acceptor is used, the enzyme of the present invention obtainedin the above does not substantially have the activity to transfer GlcUAfrom UDP-GlcUA. Based on these results, it was shown that the aboveenzyme of the present invention is not capable of transferring GlcUA tothe non-reduced terminal GlcUA but is capable of transferring oneresidue of GalNAc (or, though slight, GlcNAc).

Also, it was shown that the enzyme of the present invention obtained inthe above transfers GlcUA from UDP-GlcUA to a sugar chain (acceptor)having a chondroitin backbone whose non-reduced terminal is GalNAc.Also, it was shown that when the acceptor is used, the enzyme of thepresent invention obtained in the above substantially have no activitiesto transfer GalNAc from UDP-GalNAc and to transfer GlcNAc fromUDP-GlcNAc. Based on these results, it was shown that the above enzymeof the present invention is not capable of transferring GalNAc to thenon-reduced terminal GalNAc but is capable of transferring one residueof GlcUA.

Based on the above, it was shown that the above enzyme of the presentinvention is capable of transferring GlcUA and GalNAc alternately from aGlcUA donor and a GalNAc donor, respectively, to the sugar chainnon-reduced terminal.

(5) Analysis of Specificity of Acceptor Substrate

Using tetrasaccharide (140 pmol) or hexasaccharide (140 pmol) ofchondroitin sulfate C degraded and purified using testicularhyaluronidase, tetrasaccharide (260 pmol) or hexasaccharide (175 pmol)of chondroitin degraded and purified by the Nagasawa's method(Carbohydrate Research, 97, 263-278 (1981)), hexasaccharide (175 pmol)of hyaluronic acid degraded and purified using testicular hyaluronidase,chondroitin sulfate C (molecular weight 20,000), chondroitin (molecularweight 10,000), dermatan sulfate (molecular weight 15,000), hyaluronicacid (molecular weight 20,000) or heparin (molecular weight 10,000) asthe acceptor, the transferring activity was examined by the followingmethod. Also, the sugar chains were purchased from SeikagakuCorporation.

The enzyme of the present invention (2 μg), UDP-GalNAc (Sigma) (60pmol), UDP-GlcUA (Sigma) (0.6 nmol) and UDP-[³H]GalNAc (0.1 nmol, 0.1μCi) as the donors and each of the above sugar chains as the acceptorwere added to 50 mM Tris-HCl (pH 7.2) containing 20 mM MnCl₂, 0.1 M(NH₄)₂ SO₄ and 1 M ethylene glycol, and the total volume was adjusted to50 μl, and then the enzyme reaction was carried out at 30° C. for 30minutes and the enzyme was heat-inactivated. The reaction solution wasapplied to a Superdex Peptide column (300×φ10 mm: Amersham Biosciences,chromatography conditions; buffer: 0.2 M NaCl, flow rate: 0.5 ml/min),and the eluate was fractionated at 0.5 ml and the radioactivity (countof [³H]) of each fraction was measured using a scintillation counter.The results are shown in Table 2. The parenthesized numerals in thetable are relative values when the quantity of radioactivity (amount oftransferred GalNAc) by the use of the hexasaccharide of chondroitinsulfate C as the acceptor was defined as 100%.

TABLE 2 Specific activity of [³H] incorporation Acceptor substratenmol/min/mg protein % Chondroitin Tetrasaccharide 1.44 ± 0.24 43.0sulfate C Hexasaccharide 3.34 ± 0.50 100.0 Polysaccharide 3.41 ± 0.48100.0 (M.W. 20,000) Chondroitin Tetrasaccharide 1.12 ± 0.21 33.5Hexasaccharide 1.24 ± 0.45 37.0 Polysaccharide 0.53 ± 0.13 15.8 (M.W.10,000) Hyaluronic acid Hexasaccharide 0.80 ± 0.15 24.0 Polysaccharide0.27 ± 0.02 8.2 (M.W. 20,000) Dermatan sulfate Polysaccharide 0.06 ±0.02 1.9 (M.W. 15,000) Heparin Polysaccharide 0.0 ± 0.0 0.0 (M.W.10,000)

Based on the above results, it was shown that the hexasaccharide ofchondroitin sulfate C becomes the most suitable acceptor substrate. Thechondroitin hexasaccharide also functioned as an acceptor substrate, butits activity was low (37%) in comparison with the case of thehexasaccharide of chondroitin sulfate C. Incorporation into thechondroitin sulfate tetrasaccharide or chondroitin tetrasaccharide wasthe same (43% and 33.5%, respectively). To our surprise, the hyaluronicacid hexasaccharide and hyaluronic acid (molecular weight 20,000) alsofunctioned as acceptor substrates. The incorporation level ofchondroitin sulfate C (molecular weight 20,000) was similar to that ofthe hexasaccharide of chondroitin sulfate C. The incorporation was notso high in the case of chondroitin (molecular weight 10,000).

In summing up the above results, it was shown that the above enzyme ofthe present invention uses oligosaccharides and polysaccharides havingchondroitin backbone (at least tetrasaccharide, hexasaccharide andheptasaccharide of chondroitin sulfate C, chondroitin tetrasaccharideand hexasaccharide, chondroitin sulfate C (molecular weight 20,000) andchondroitin (molecular weight 10,000)) and oligosaccharides andpolysaccharides of hyaluronic acid (at least hyaluronic acidhexasaccharide and hyaluronic acid (molecular weight 20,000)) asacceptors.

On the other hand, incorporation of the radioactivity was not found indermatan sulfate (molecular weight 15,000) and heparin (molecular weight10,000), showing that they do not substantially function as acceptorsubstrates.

(6) Analysis of Influence by Temperature

The enzyme reaction was carried out in the same manner as the above “(2)Analysis of enzyme activity” by changing the enzyme reaction temperatureto 25, 30, 37, 40, 45 or 100° C., and the solution after the reactionwas applied to a Superdex 75 column (300×φ10 mm: Amersham Biosciences,chromatography conditions; buffer: 0.2 M NaCl, flow rate: 0.5 ml/min).The eluate was fractionated in 1 ml portions and the radioactivity(count of [³H]) of each fraction was measured using a scintillationcounter. The results are shown in FIG. 6. Also, the lozenge, square,triangle, x and * marks in FIG. 6 show the results of 25, 30, 37, 40, 45and 100° C., respectively. Also, the circle in FIG. 6 shows the resultof a control (wherein heat-inactivated enzyme of the present inventionwas used).

As shown in FIG. 6, under the reaction conditions and within thetemperature range examined this time, the molecular weight of thereaction product became small as the temperature was increased. Thehighest incorporation was found at 30° C., but the product having thelargest molecular weight was obtained at, 25° C.

It is considered from the results that enzyme activity of the aboveenzyme of the present invention decreases as the reaction temperatureincreases starting at 25° C. under the reaction conditions of this time.

(7) Analysis of Influence by Metal Ions and the Like

The enzyme reaction was carried out in the same manner as the above “(2)Analysis of enzyme activity”, except that each of various metal salts(MnCl₂, FeCl₂, MgCl₂, CaCl₂ or CuCl₂) or EDTA was added instead ofMnCl₂, and the solution after the reaction was applied to a Superdex 75column (300×φ10 mm: Amersham Biosciences, chromatography conditions;buffer: 0.2 M NaCl, flow rate: 0.5 ml/min). The eluate was fractionatedat 1 ml and the radioactivity (count of [³H]) of each fraction wasmeasured using a scintillation counter. Relative values when theradioactivity in addition of MnCl₂ was defined as 100% are shown below.

TABLE 3 Metal salt Relative value of radioactivity (%) MnCl₂ 100.0 FeCl₂30.6 MgCl₂ 30.7 CaCl₂ 0.0 CuCl₂ 0.0 EDTA 0.0

Based on the results; the above enzyme of the present invention showedthe highest activity in the presence of Mn²⁺ ion. Also, in the presenceof Fe²⁺ or Mg²⁺ ion, it showed about 30% of the activity in comparisonwith the case of the presence of Mn²⁺ ion. Also, it was shown that itdoes not substantially act in the presence of Ca²⁺ or Cu²⁺ ion orethylenediaminetetraacetic acid.

In addition, it was shown that the above enzyme of the present inventionacts in the presence of Fe²⁺ or Mg²⁺ ion too, and its enzyme activity inthe presence of Mn²⁺ ion is higher than the enzyme activity in thepresence of Fe²⁺ or Mg²⁺ ion.

(8) Optimum Reaction pH

When optimum reaction pH of the enzyme of the present invention wasexamined by changing the pH of the above “(2) Analysis of enzymeactivity” to various levels, it was from pH 7.0 to 7.5 .

(9) Relation with Enzyme Reaction Time

By setting the enzyme reaction time to 10 minutes, 30 minutes, 1 hour, 3hours, 6 hours or 18 hours, the enzyme reaction was carried out in thesame manner as the above “(2) Analysis of enzyme activity”, and theradioactivity incorporated into the enzyme reaction product wasanalyzed. Gel filtration patterns of [³H]GalNAc after various reactionperiods are shown in FIG. 7, and the total amounts of the incorporationof radioactivity after various enzyme reaction periods in FIG. 8. Theopen circle, closed circle, open triangle, closed triangle, open squareand closed square show the results after 10 minutes, 30 minutes, 1 hour,3 hours, 6 hours and 18 hours, respectively. Also, the arrow with “20k”, “10 k”, “5 k”, “14”, “8” or “6” shows the elution position ofmolecular weight 20,000, 10,000 , 5,000, tetradecasaccharide (molecularweight: about 2,800), octasaccharide (molecular weight: about 1,600) orhexasaccharide (molecular weight: about 1,200) of hyaluronic acid(standard), respectively.

It was shown from the results of FIG. 8 that under the test conditions,quick incorporation of [³H]GalNAc is found after 3 hours and 6 hours,but the incorporation becomes slow as the reaction draws close to 20hours.

Also, from the results of FIG. 7, it was shown that the incorporationincreases and a reaction product of high molecular weight is obtainedafter a long period of reaction time.

In addition, a high molecular weight product was quickly obtained whenan acceptor substrate (hexasaccharide) was set to a lower concentration,and a low molecular weight product was obtained when the acceptorsubstrate (hexasaccharide) was set to a high concentration.

(10) Determination of Michaelis Constant (Km)

The radioactivity incorporated into the enzyme reaction product wasmeasured in accordance with the above “(2) Analysis of enzyme activity”,by setting using amount of the enzyme of the present invention to 1.3μg, containing the one donor substrate (UDP-sugar; UDP-GlcUA orUDP-GalNAc) in a fixed concentration (240 μM), and adding thereto theother radio-labeled donor substrate (radiation UDP-sugar; UDP-[³H]GalNAcor UDP-[¹⁴C]GlcUA) having various (0.6 to 200 μM). Independent testswere carried out three times, and the average value was used as themeasured value.

Relationship between the incorporated radioactivity (V) and substrateconcentration (S) of UDP-sugar is shown in FIG. 9, and its doublereciprocal plot in FIG. 10. Closed circle and open square in thedrawings show results on UDP-GlcUA and UDP-GalNAc, respectively.

As the result, the Km for UDP-GlcUA and the Km for UDP-GalNAc werecalculated to be 3.44 μM and 31.6 μM, respectively.

While the present invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofskill in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof. Allreferences cited herein are incorporated in their entirety.

This application is based on Japanese application Nos. 2001-244685,2001-324127 and 2002-103136 filed on Aug. 10, 2001, Oct. 22, 2001 andApr. 5, 2002, the entire contents of which are incorporated hereinto byreference.

1-17. (canceled)
 18. A method for producing a sugar chain comprising:contacting an acceptor sugar chain with at least one GalNAc donor,GlcNAc donor, or GlcUA donor and an isolated protein that haschondroitin polymerase activity; wherein said isolated protein that haschondroitin polymerase activity comprises SEQ ID NO: 2, a fragment ofSEQ ID NO: 2, or an amino acid sequence identical to SEQ ID NO: 2 exceptthat it has 1 to 30 of its amino acid residue(s) deleted, orsubstituted, or 1 to 30 amino acid residues inserted within the sequenceof SEQ ID NO: 2; wherein said isolated protein is not membrane-hound.19. The method of claim 18 that produces a sugar chain represented bythe formula (3):GalNAc-GlcUA-X—R¹  (3); comprising: contacting the protein that haschondroitin polymerase activity with a GalNAc donor and an acceptorsugar chain represented by the formula (1):GlcUA-X—R¹  (1); wherein GlcUA represents D-glucuronic acid; GalNAcrepresents N-acetyl-D-galactosamine; X represents GalNAc or GlcNAc inwhich GlcNAc represents N-acetyl-D-glucosamine;-represents a glycosidiclinkage; and R¹ represents an any group.
 20. The method of claim 18 thatproduces a sugar chain represented by formula (4):GlcNAc-GlcUA-X—R¹  (4); comprising: contacting the isolated proteinhaving chondroitin polymerase activity with a GlcNAc donor and anacceptor sugar chain represented by the formula (1):GlcUA-X—R¹  (1); wherein GlcUA represents D-glucuronic acid; GlcNAcrepresents N-acetyl-D-glucosamine; X represents GalNAc or GlcNAc inwhich GalNAc represents N-acetyl-D-galactosamine;-represents aglycosidic linkage; and R¹ represents an any group.
 21. The method ofclaim 18 that produces a sugar chain represented by formula (5):GlcUA-GalNAc-GlcUA-R²  (5); comprising: contacting the isolated proteinhaving chondroitin polymerase activity with a GlcUA donor and anacceptor sugar chain represented by the formula (2):GalNAc-GlcUA-R²  (2) wherein GlcUA represents D-glucuronic acid; GalNAcrepresents N-acetyl-D-galactosamine;-represents a glycosidic linkage;and R² represents an any group.
 22. The method of claim 18 that producesa sugar chain selected from the group consisting of:(GlcUA-GalNAc)n-GlcUA-X—R¹  (6) andGalNAc-(GlcUA-GalNAc)n-GlcUA-X—R¹  (8): comprising: contacting theisolated protein having chondroitin polymerase activity with a GalNAcdonor, a GlcUA donor and an acceptor sugar chain represented by formula(1):GlcUA-X—R¹  (1); wherein GlcUA represents D-glucuronic acid; GalNAcrepresents N-acetyl-D-galactosamine; X represents GalNAc or GlcNAc inwhich GlcNAc represents N-acetyl-D-glucosamine;-represents a glycosidiclinkage; R¹ represents an any group; and n is an integer of 1 or more.23. The method of claim 18 that produces a sugar chain selected from thegroup consisting of:(GalNAc-GlcUA)n-GalNAc-GlcUA-R²  (7) andGlcUA-(GalNAc-GlcUA)n-GalNAc-GlcUA-R²  (9); comprising: contacting theisolated protein having chondroitin polymerase activity with a GalNAcdonor, a GlcUA donor and an acceptor sugar chain represented by formula(2):GalNAc-GlcUA-R²  (2); wherein GlcUA represents D-glucuronic acid; GalNAcrepresents N-acetyl-D-galactosamine;-represents a glycosidic linkage; R²represents an any group; and n represents an integer of 1 or more. 24.The method of claim 18, wherein said isolated protein comprises SEQ IDNO: 2 and has chondroitin polymerase activity.
 25. The method of claim18, wherein said isolated protein consists of a fragment of SEQ ID NO: 2that has chondroitin polymerase activity.
 26. The method of claim 18,wherein said isolated protein comprises a sequence identical to SEQ IDNO: 2 except that it has 1 to 30 of its amino acid residue(s) deleted,substituted, or 1 to 30 amino acid residues inserted within the sequenceof SEQ ID NO: 2; and wherein said isolated protein has chondroitinpolymerase activity.
 27. The method of claim 18, wherein said contactingoccurs in the presence of at least one of Mn²⁺, Fe²⁺, or Mg²⁺.
 28. Themethod of claim 18, comprising contacting said acceptor sugar chain withnucleoside diphosphate-GlcUA and/or UDP-GlcUA.
 29. The method of claim18, comprising contacting said acceptor sugar chain with a nucleosidediphosphate-GalNAc and/or UDP-GalNAc.
 30. The method of claim 18,wherein said acceptor sugar chain has a chondroitin backbone.
 31. Themethod of claim 18, wherein said acceptor sugar chain is anoligosaccharide.
 32. The method of claim 18, comprising contacting anacceptor sugar chain that has a hyaluronic acid backbone (a disacchariderepeating structure of GlcUA residue and GlcNAc residue) with a GalNAcdonor.